Distinct membrane compartments have clearly distinct populations of membrane and soluble proteins. For this asymmetry within the cell to be established and retained, specific macromolecules must be segregated from bulk proteins and packaged into specialized vesicles that transfer them to the appropriate membrane, where they deliver their cargo by fusion. The clathrin-dependent pathway is the prototypical sorting pathway, and is the focus of this grant. The central figures in clathrin coat formation and cargo capture are the AP complexes: AP-1, responsible for traffic at the TGN, AP-2, responsible for traffic at the plasma membrane. We propose to study how APs recognize, capture and target cargo by determining how the sequence of the sorting signals present in the cytoplasmic domain of the cargo proteins influences recognition, by studying the regulation of recognition, and by determining the molecular structure of the interface between the sorting signal and the AP complex. AP-3 is a complex related to AP-1 and AP-2 that is suspected to be involved in TGN sorting but not yet known to interact with clathrin. We will determine whether it uses the same methods for sorting signal recognition and cargo capture as the other AP complexes. We will also study the second important interface known to be formed by AP-1 and AP-2, the interface with clathrin: we have produced diffracting crystals of the relevant domain of clathrin, and we will determine its structure alone and in complex with the relevant portion of the AP complex. We thus hope to provide a molecular picture of the key steps in sorting and coat assembly.